JPS61146189A - Novel antibiotic substance af-7368e and preparation thereof - Google Patents

Abstract

NEW MATERIAL:The antibiotic substance AF-7368E having the following physical and chemical properties. Elemental analysis (%), C 57.85, H 8.10, N 2.06; melting point, no definite melting point, coloring gradually from 130 deg.C and browing and decomposing at 150-160 deg.C; [alpha]D=+87.4 deg. (C=1.0, methanol); solubility, easily soluble in methanol, ethanol, butanol and dimethyl sulfoxide, soluble in chloroform and methylene chloride, hardly soluble in water, acetone, acetonitrile and ethyl acetate, insoluble in hexane, carbon tetrachloride, ether and benzene; reactivity, positive to ninhydrin reaction and alpha-naphthol reaction, negative to Sakaguchi's reaction, Millon reaction, and Elson-Morgan reaction; nature, acidic; color, white, etc. USE:Antibacterial agent. PREPARATION:A microorganism belonging to Sterptomyces genus [preferably Sterptomyces hygroscopicus No.7368 (FERM P-7440)] is cultured at 25-35 deg.C for 2-5 days preferably under nearly neutral condition.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a novel antibiotic AF-7368E substance and its production method. More specifically, the present invention relates to an antifungal antibiotic AF-7368E substance that specifically acts on molds, yeasts, etc., and a method for producing the substance using a microorganism belonging to the genus Streptomyces.

BACKGROUND OF THE INVENTION The present inventors previously filed a patent application for an antifungal antibiotic substance AF-7368A produced by a microorganism belonging to the genus Streptomyces (Japanese Patent Application No. 59-29127).

Problems to be Solved by the Invention Although the aforementioned AF-7368A substance itself has strong antifungal activity, the present invention aims to provide a substance that has even stronger antifungal activity. shall be.

Means to Solve the Problems The present inventors isolated microorganisms from various types of soil samples and searched for the antibiotics they produced, and found that certain microorganisms were specific to mold and yeast. I learned that they produce a wide variety of effective antifungal antibiotics. Among these substances, the AF-7368A substance is as described above, but subsequently we succeeded in isolating a second effective substance with clearly different physical and chemical properties from this AF-7368A substance, and this substance was named AF-7368E. Named substance.

The antibiotic AF-7368E substance of the present invention is a novel anti-biotic having the following physicochemical and biological properties.

It is a biological substance.

■Elemental analysis: Carbon 57.85% Hydrogen 8.10% Nitrogen 2.06% ■Melting point: Does not show a clear melting point, gradually changes color from 130°C, and browns and decomposes at 150-160°C.

■ Specific optical rotation: (α), +87.4° (C = 1.0, methanol) ■ Ultraviolet absorption spectrum (in methanol) = 232 n
It has an absorption maximum near m. The absorption maximum does not shift even in alkaline methanol and acidic methanol solutions (
(shown in Figure 1).

■Infrared absorption spectrum (KBr tablet method): Shown in Figure 2.

■Solubility in solvents: Easily soluble in methanol, ethanol, butanol, dimethyl sulfoxide; chloroform.

Soluble in methylene chloride; water, acetone, acetonitrile,
Slightly soluble in ethyl acetate; hexane, carbon tetrachloride, ether,
Insoluble in benzene.

■Color reaction: Positive for ninhydrin reaction, α-naphthol reaction, negative for Sakaguchi reaction, Miron reaction, Elson-Morgan reaction.

■Distinction between acidic, neutral, and basic: Acidic ■Substance color: White [Phase] Retention time in high performance liquid chromatography: TS
Kge 1-ODS-120T column (manufactured by Toyo Soda Co., Ltd.,
The retention time in high performance liquid chromatography using 4.6fiφX 250mm is 16.0 to 18.0 minutes.

(Solvent: 5% methanol, flow rate: 1-7 minutes) ■ Rf value in thin layer chromatography Using Nisilica gel thin layer chromatography (manufactured by Merck; Kieselgel 60), n-butanol:acetic acid:water (4:1: The Rf value when expanded in 1) is 0.39.

@Antibacterial spectrum: The minimum inhibitory concentrations against various microorganisms measured by the agar dilution method are shown in Table 1. As can be seen from the table, it exhibits a growth inhibiting effect on molds and yeasts, but does not act on bacteria within the tested range.

Table 1 When the physicochemical properties and biological properties of the above-mentioned antibiotic AF-7368E substance were compared with those of known antibiotics, no corresponding substance was found, and this substance was confirmed to be a new antibiotic. Furthermore, the antifungal activity of the substance of the present invention is
The minimum inhibitory concentration against certain fungi is, for example, several tens of tenths of a fraction of a fraction of a fraction of a fraction of that of the aforementioned AF-7368A substance.

Antibiotic AF-7368E substance is Aspergillus (As
pergillus) genus, Rhizopus (Rh1zop)
Trfchophyto
n) Genus, Pyricularia
It has antibacterial activity against molds and yeasts of the genus Geotricum, Geotricum, and Cryptococcus, so it is used as an agricultural antibacterial agent or as a preventive or therapeutic agent for mycoses in animals and humans. Is possible.

As an example of the microorganism used to produce the antibiotic AF-7368E substance according to the present invention, the 1m7368 strain belonging to the genus Streptomyces, which the present inventors isolated from soil in Hagi City, Yamaguchi Prefecture, is cited. It will be done. llh 7
The mycological properties of strain 368 are as follows.

(a) Aerial hyphae of around 1 μm extend from the well-branched basal hyphae, and simply branch from the main axis to form side branches, with chains of spiral spores seen at the tips. In the late stage of cultivation, a moist black area (hygroscopic area) appears on the aerial mycelium. Flagellated spores, sclerotia, and sporangia are not observed. The surface structure of the spore is smooth. The shape of the spores is oval to cylindrical, and the number of chains is 10 or more. Spore size is 0.6~
0.8 x 0.9 to 1.4.

(bl) Growth status in each medium Table 2 shows the growth status when cultured for 14 to 21 hours.

(Left below) (C1 Physiological properties ■ Growth temperature range: Bennetti's brot
As a result of a growth test in a temperature gradient culture device using h.
Grows in the range ~38°C.

■ Liquefaction of gelatin: Positive ■ Hydrolysis of starch: Positive ■ Coagulation of skim milk: Negative ■ Peptonization of skim milk: Positive ■ Production of melanin-like pigments: Negative (d> Assimilation of various carbon sources (Pridham-Godelive agar) on the medium) Assimilates glucose, fructose, galactose, raffinose, mannitol, inositite, and sucrose.

Does not assimilate xylose, arabinose, rhamnose, and salicin.

From the above, the mycological characteristics of the lb 7368 strain are summarized as follows:
Aerial hyphae form spiral threads, and the spore surface structure is smooth. The color of the aerial mycelium is gray, and hygroscopic areas are observed in the late stage of cultivation. The underside is yellowish white to yellowish brown, with no special color tones.

For bacterial species exhibiting the above mycological properties, purge aids
Bergey's Manual of Det.
erminative Bacteriology)
7th edition (1957), 8th edition (1974), Schirling and Godlieb's 15P (International Edition)
Streptomyces Project Reports (1968, 1969 and 1972) and Waxman, The Actinomycetes
etetes) Volume 2 (1961), Streptomyces bygroscopicus (Strepton+y
ces hygroscopiccus).

That is, the basic properties of (1) forming spiral threads, (2) epiphyting gray aerial hyphae, and (3) hygrocopic aerial hyphae are the same. However, there are some differences in sugar assimilation. Therefore, N17
Although strain 368 has some differences from Streptomyces bigroscopicus, the difference is not large enough to be considered a different bacterial species, and it was determined that the strain is the same species as Streptomyces bigroscopicus. Mysse
Vigroscopicus m7368 (Streptomy
ces hygroscopicuslm 7368)
It was named. This strain was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, as part of the FERMP-7440 (FERMP-7440)
It has been deposited as.

Streptomyces pygroscopicus strain 7368 as a microorganism used in the present invention is one example, and mutant strains obtained by mutating this strain by means using ultraviolet rays, radiation, chemical agents, etc. Antibiotic AF-7368 is a microorganism belonging to the genus Streptomyces.
Any microorganism capable of producing substance B can be used in the present invention.

To produce the antibiotic AF-7368E substance by the method of the present invention, first, a strain producing the antibiotic AF-7368E substance belonging to the genus Streptomyces is cultured in a medium, and the antibiotic AF-7368E substance is added to the culture. Let it accumulate.

As the culture method, a general culture method for actinomycetes is used.

Examples of the culture medium include peptone, meat extract, yeast extract, soybean flour, cottonseed flour, cornstarch liquor, nitrogen sources such as sodium nitrate, ammonium nitrate, and ammonium sulfate, and carbon sources such as glucose, starch, sucrose, and dextrin. Inorganic salts such as phosphate, potassium, sodium, magnesium, calcium, zinc, iron, etc. are used as necessary.

The culturing method is preferably carried out aerobically with aeration and stirring. Culture conditions such as pH 1 of the medium, culture temperature, and culture time are selected to be suitable for the growth of the bacterial strain and to maximize the production of the antibiotic AF-7368E substance. For example, p of the medium
H is preferably 5 to 8, particularly preferably around neutrality, and the culture temperature is preferably 25 to 35°C. Culture time is antibiotic AF-
7368E@IK.

All you have to do is choose the time when the maximum accumulation of microorganisms occurs, which is usually 2 to 5 days.Conditions such as medium composition, pH 1 of the medium, culture temperature, culture time, and aeration/agitation depend on the type of strain used and external conditions. It goes without saying that the optimal conditions are selected by such methods.

Antibiotic AF-73 was extracted from the culture thus obtained.
The 68E substance can be obtained by appropriately utilizing purification means commonly used for antibiotic purification. For example, methods that utilize differences in adsorption affinity, methods that utilize differences in molecular weight, methods that utilize differences in ionic bond strength, and methods that utilize solubility in solvents are used. Alternatively, they may be performed in combination or repeatedly.

- To explain in more detail by way of example, the antibiotic AF-7
To separate the 368E substance from the culture, first, filtration,
Fractionate into clear filtrate and bacterial cells using centrifugation or other means. Usually, the substance of the present invention is applied to the bacterial cell compartment more than the filtrate compartment.
It is present in ~4 times as much amount, but depending on the culture conditions, the proportion present in the filtrate section may be high. To collect the substance of the present invention from the bacterial cell section, add methanol, ethanol,
Extract by adding a solvent such as propatool. In addition, in order to collect the present invention substance from the filtrate section, use the Diaion HP
-20 (manufactured by Mitsubishi Chemical Corporation), Amberlight XAD-2 (
After the substance of the present invention is adsorbed onto a synthetic adsorbent such as (manufactured by Rohm & Haas) or an adsorbent such as activated carbon, it is eluted with a solvent such as methanol, ethanol, or propatool.

The extract obtained from the bacterial cell section and the eluate obtained from the filtrate section by adsorption with an adsorbent and elution are combined and concentrated under reduced pressure at 50°C or below to remove the solvent. Obtain a concentrated solution of the invention substance. Then, 1~
Two times the amount of n-butanol is added to extract the substance of the present invention into the n-butanol layer. Water-soluble impurities in the n-butanol layer are removed to the aqueous layer by adding water and shaking and extracting.

The n-butanol layer is obtained by adding a solvent such as acetone or ethyl acetate to it, or by concentrating it to dryness and then dissolving it in a solvent such as methanol, followed by normal phase chromatography using silica gel or the like. By concentrating the obtained active fraction to dryness, a crude sample of the substance of the present invention can be obtained. After dissolving this crude sample in a solvent such as ethanol, high-purity AF- 7368B material is obtained.

Applications - Sucrose 2%, Polypeptone 0.5%, Meat extract 0.3%, Yeast extract 0.1%, Potassium phosphate 0.
1%, magnesium sulfate 0.05% (pH 6,8) 100 if! 500- with
Prepare a Yonosaka Lough flask and add Streptomyces bigroscopicus 1IkL7368 (FERM
P-7440) was inoculated and cultured with shaking at 28°C for 2 days to obtain a seed culture. Next, put 2ON of a medium with the same composition as above into a 30β volume fermentation tank, and add 200mf of the above seed culture solution.
! After inoculation, culture was carried out for 66 hours at a stirring speed of 250 rpm, an aeration rate of 101/min, an internal pressure of 0.5 kg/cj, and a temperature of 28°C.

The culture solution was filtered to obtain 181 filtrate and 230 g of wet bacterial cells. The filtrate was passed through a Diaion HP-20 column (bed volume: 1Jl, 800ml!) to adsorb the substance of the present invention, and then passed through 2.5ml of 50% methanol solution and 2.5ml of pure methanol in order for elution. The active fraction was eluted into the pure methanol portion to obtain eluate 1.51.On the other hand, for the wet bacterial cells, after adding 21 methanol and stirring for 2 hours, the bacterial cells were removed and eluate 1.51 was obtained. I got .8E.

This extract and the eluate of the active fraction from Diaion HP-20 were combined and concentrated under reduced pressure to obtain a brown syrup solution. This was suspended in 500ml of water, 1.5 times the volume of n-butanol was added, and the suspension was shaken and stirred to extract both active components into the n-butanol layer. The n-butanol layer was separated, and the same volume of water was added thereto and washed by shaking.Then-butanol layer was separated, and an equal volume of acetone was added thereto. Next, silica gel (trade name, manufactured by Wako Pure Chemical Industries, Ltd.) was equilibrated in advance with a mixture of n-butanol and acetone (1:1).

A column packed with 200 g of Wakogel C-200) was prepared, and an n-butanol-acetone solution containing the above active fraction was passed therethrough to adsorb the activity. Thereafter, the column was washed with a 2N solvent of the same composition, and then eluted with a mixture of methanol and ethyl acetate (4:1) 2β to obtain an eluate 800- containing an active fraction. This eluate was concentrated to dryness under reduced pressure to obtain 450 ml of yellow powder. This yellow powder is 501n1
After dissolving in ethanol of
The mixture was loaded on a microtube and eluted with a solvent of the same composition to obtain a 1°21 eluate containing the active fraction. After adding 1.2 mm of purified water to this, prepare a PrepPAK-500/C1B cartridge column (manufactured by Waters; 5CIllφX30C1) equilibrated with a 30% ethanol solution
1) to adsorb the substance of the present invention. Solvent 5 with the same composition
The column was washed with 00- followed by elution with a 60% ethanol solution.

The activity eluted between 1.312 and 2.81.

This active fraction was concentrated to dryness under reduced pressure, then dissolved in 1 g of 30% ethanol solution, and adsorbed onto the same column again.

After washing the column with 30% ethanol solution 500- and eluting with 75% methanol solution, the activity was 2.
It was eluted in both fractions from Ol to 3.51. This eluate was concentrated under reduced pressure and then lyophilized to obtain 32 ml of pure antibiotic substance AF-7368E.

Effects of the Invention According to the present invention, firstly, a novel antifungal antibiotic having antibacterial activity against molds and yeasts such as Aspergillus, Rhizobus, Trichocyton, Piricularia, Geotrichum, and Cryptococcus spp. Material AF-7368
E substance is provided.

Second, AF-7368E belongs to the genus Streptomyces.
A method for producing the above substance using a microorganism capable of producing the substance is provided. The antibacterial activity of the AF-7368E substance has a minimum inhibitory concentration against specific fungi of several tenths of a fraction of a fraction of a second, compared to the AF-7368A substance previously discovered by the present inventors.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the ultraviolet absorption spectrum (in methanol) of the antibiotic AF-7368E substance of the present invention, and FIG. 2 is a diagram showing the same infrared absorption spectrum (KBr tablet method).

Claims (1)

[Claims] 1. A novel antibiotic AF-73 having the following physical and chemical properties:
68E substance. (1) Elemental analysis: 57.85% carbon, 8.10% hydrogen, 2.06% nitrogen (2) Melting point: Does not show a clear melting point, gradually changes color from 130°C, and browns and decomposes at 150-160°C. (3) Specific rotation: [α]_D+87.4° (C=1.0, methanol) (4) Ultraviolet absorption spectrum (in methanol): 232
It has an absorption maximum near nm (shown in Figure 1). (5) Infrared absorption spectrum (KBr tablet method): Shown in FIG. (6) Solubility in solvents: Easily soluble in methanol, ethanol, butanol, dimethyl sulfoxide; Soluble in chloroform, methylene chloride; Slightly soluble in water, acetone, acetonitrile, ethyl acetate; In hexane, carbon tetrachloride, ether, benzene Insoluble. (7) Color reaction: Positive for ninhydrin reaction, α-naphthol reaction, negative for Sakaguchi reaction, Millon reaction, Elson-Morgan reaction (8) Distinction between acidic, neutral, and basic: Acidic (9) Color of substance: White 2 Belongs to the genus Streptomyces and is an antibiotic AF-736
A method for producing a novel antibiotic AF-7368E substance, which comprises culturing a microorganism capable of producing an 8E substance in a medium to produce and accumulate an antibiotic AF-7368E substance, and collecting the same. 3. The method for producing a novel antibiotic AF-7368E substance according to claim 2, wherein the microorganism belonging to the genus Streptomyces is Streptomyces hygroscopicus. 4 Streptomyces hygroscopicus is Streptomyces hygroscopicus no. 7368 (F
A method for producing the novel antibiotic AF-7368E substance according to claim 3, which is ERM P-7440).